Rifampin In Mycobacterium Tuberculosis Research Articles

The role of Xpert MTB/RIF using bronchoalveolar lavage fluid in active screening: insights from a tuberculosis outbreak in a junior school in eastern China.

Tuberculosis (TB) outbreaks in schools present a public health challenge. In order to effectively control the spread of transmission, timely screening, accurate diagnosis and comprehensive epidemiological investigations are essential. In July 2021, a TB outbreak occurred in a junior high school in Y City, Zhejiang Province. Students and faculty were screened for TB by symptom screening, chest radiography, and tuberculin skin test during four rounds of contact screenings. For sputum smear-negative and sputum-scarce patients, bronchoscopy was used to collect BAL samples for Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF). Whole-genome sequencing and bioinformatics analysis were performed on isolates to identify the strains of MTB isolates and predict drug resistance. Between July 2021 and November 2021, a total of 1,257 students and faculty were screened for TB during screenings. A total of 15 students (1.2% of persons screened) aged 15 years were diagnosed with TB. Eighty percent (12/15) of the cases were laboratory-confirmed (10/12 [83%] Xpert MTB/RIF-positive, 2/12 [17%] culture-positive). Most cases (12/15 [80%]) were in students from Class 2. All cases were asymptomatic except for the index case who had symptoms for more than two months. Seven MTB isolates were collected and belonged to lineage 2. Our findings demonstrated the potential of Xpert MTB/RIF using BAL as a screening tool in school TB outbreaks for sputum smear-negative and sputum-sparse suspects, which may not only rapidly improves diagnostic accuracy, but also facilitates epidemiological investigations and homology analysis.

Early Microbiologic Markers of Pulmonary Tuberculosis Treatment Outcomes.

Rationale: Earlier biomarkers of pulmonary tuberculosis (PTB) treatment outcomes are critical to monitor shortened anti-TB treatment (ATT). Objectives: To identify early microbiologic markers of unfavorable TB treatment outcomes. Methods: We performed a subanalysis of 2 prospective TB cohort studies conducted from 2013 to 2019 in India. We included participants aged ⩾18 years who initiated 6-month ATT for clinically or microbiologically diagnosed drug-sensitive PTB and completed at least one follow-up visit. Sputum specimens were subjected to a baseline Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay, acid-fast bacilli (AFB) microscopy and liquid and solid cultures, and serial AFB microscopy and liquid and solid cultures at weeks 2, 4, and 8. Poisson regression was used to assess the impact of available microbiologic markers (test positivity, smear grade, time to detection, and time to conversion) on a composite outcome of failure, recurrence, or death by 18 months after the end of treatment. Models were adjusted for age, sex, nutritional status, diabetes, smoking, alcohol consumption, and regimen type. Results: Among 1,098 eligible cases, there were 251 (22%) adverse TB treatment outcomes: 127 (51%) treatment failures, 73 (29%) recurrences, and 51 (20%) deaths. The primary outcome was independently associated with the Xpert MTB/RIF assay (medium-positive adjusted incidence rate ratio [aIRR], 1.91; 95% confidence interval [CI], 1.07-3.40; high-positive aIRR, 2.51; 95% CI, 1.41-4.46), positive AFB smear (aIRR, 1.48; 95% CI, 1.06-2.06), and positive liquid culture (aIRR, 1.98; 95% CI, 1.21-3.23) at baseline; Week 2 positive liquid culture (aIRR, 1.47; 95% CI, 1.04-2.09); and Week 8 positive AFB smear (aIRR, 1.63; 95% CI, 1.06-2.50) and positive liquid culture (aIRR, 1.54; 95% CI, 1.07-2.22). There was no evidence of Mycobacterium tuberculosis growth in the Mycobacterium Growth Indicator Tube at Week 4 conferring a higher risk of adverse outcomes (aIRR, 1.25; 95% CI, 0.89-1.75). Conclusions: Our analysis identifies Week 2 respiratory mycobacterial culture as the earliest microbiologic marker of unfavorable PTB treatment outcomes.

Utility of laboratory diagnostic tests in women suspected of genital tuberculosis attending a tertiary care teaching hospital.

Genital Tuberculosis is a form of extrapulmonary tuberculosis, which if not diagnosed early can lead to complications. The objective of this study was to determine the sensitivity and specificity of Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay in genital tuberculosis (TB) in comparison with culture as a gold standard. The results of the Xpert MTB/RIF assay performed from January 2020 to August 2021 were compared with the results of culture by Mycobacterium Growth Indicator Tube (MGIT) 960. Out of 75 specimens, fluorescent microscopy and liquid culture using MGIT and Xpert assay were positive in 3 (4%), 21 (28%), and 14 (18%), respectively. The sensitivity and specificity of the Xpert MTB/RIF assay were 66.67% and 100%. All smear-positive specimens were positive by culture and Xpert assay. Three specimens were positive by all the tests, i.e., microscopy, culture, and Xpert assay. Fifty-four specimens were negative by microscopy, culture, and Xpert assay. Discordance between the results of culture and Xpert assay was observed in seven specimens which were culture positive and Xpert assay negative. Three (21.42%) out of 21 culture-positive specimens showed monoresistance to rifampicin by Xpert MTB/RIF assay and culture drug susceptibility testing. Xpert MTB/RIF assay showed good sensitivity and specificity compared to liquid culture in genital TB. This test is easy to perform, provides results in 2 h, and can also detect rifampicin resistance, which is a surrogate marker for multidrug-resistant TB. Hence, the Xpert assay can be used under the National TB Elimination Program for early and rapid diagnosis of TB in endometrial specimens to prevent complications like infertility.

Xpert MTB/RIF Ultra outperformed the Xpert assay in tuberculosis lymphadenitis diagnosis: a prospective head-to-head cohort study

ObjectivesXpert Mycobacterium tuberculosis/rifampin (MTB/RIF) Ultra (Xpert-Ultra) has shown better sensitivity in comparison with Xpert MTB/RIF (Xpert) in extrapulmonary tuberculosis (TB), whereas the head-to-head comparison of these methods in TB lymphadenitis had barely been performed. MethodsPatients with undiagnosed lymphadenopathy were recruited prospectively and consecutively, and fine-needle aspiration (FNA) biopsy or lymph node tissue was collected. The specimen was subjected to smear, culture, Xpert, and Xpert-Ultra assays. Culture and/or smear for acid-fast bacilli (AFB) or AFB observed on histopathology were performed as a reference. ResultsA total of 106 participants were recruited, including 41 confirmed TB, 33 probable TB, and 32 non-TB lymphadenopathies. The head-to-head comparison for MTB detection showed that Xpert-Ultra produced the highest sensitivity when compared with smear, culture, and Xpert (75.7% vs 5.4 %, 13.5%, and 48.7%). When Xpert-Ultra outcomes were integrated for diagnosis, the percentage of confirmed TB lymphadenitis cases increased from 55.4% (41/74) to 85.1% (63/74). The sensitivities of Xpert-Ultra and Xpert on tissue were 73.6% (95% CI: 59.4-84.3) and 39.6% (95% CI: 26.8-54.0), respectively. The sensitivity of Xpert-Ultra on FNA samples (81.0%, 95% CI: 57.4-93.7) was higher than that of Xpert (71.4%, 95% CI: 47.7-87.8). ConclusionXpert-Ultra detected significantly more TB lymphadenitis cases than Xpert or culture. This superiority was particularly distinct using lymph node tissue than FNA detection.

Assessing the utility of the Xpert Mycobacterium tuberculosis/rifampin assay for analysis of bronchoalveolar lavage fluid in patients with suspected pulmonary tuberculosis

BackgroundThere is limited research assessing the utility of the Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay for the analysis of bronchoalveolar lavage fluid (BALF) in Chinese patients with suspected pulmonary tuberculosis (PTB). Thus, our objective was to determine the diagnostic accuracy of the Xpert MTB/RIF assay and evaluate its utility for the determination of rifampicin resistance.MethodsWe retrospectively analyzed BALF from 214 patients with suspected PTB between January 2018 and March 2019. Using mycobacterial culture or final clinical diagnosis as the reference standard, the diagnostic accuracy of the smear microscopy (SM), tuberculosis bacillus DNA (TB‐DNA), Xpert MTB/RIF assay, and the determination of rifampicin resistance based on the Xpert MTB/RIF assay were compared.ResultsAs compared to mycobacterial culture, the sensitivity of the Xpert MTB/RIF assay, SM, and TB‐DNA were 85.5% (74.2%–93.1%), 38.7% (26.6%–51.9%), and 67.7% (54.7%–79.1%), respectively. As compared to the final diagnosis, the specificity of the Xpert MTB/RIF assay, SM, and TB‐DNA were 100.0% (95.9%–100.0%), 94.3% (87.1%–98.1%), and 98.9% (93.8%–100.0%), respectively. The sensitivity and specificity of the rifampicin resistance detection using the Xpert MTB/RIF assay were 100% and 98.0%, respectively, with liquid culture as the reference.ConclusionsThis study demonstrates that the analysis of BALF with the Xpert MTB/RIF assay provides a rapid and accurate tool for the early diagnosis of PTB. The accuracy of diagnosis was superior compared with the SM and TB‐DNA. Moreover, Xpert is a quick and accurate method for the diagnosis of rifampicin‐resistant tuberculosis and can also provide more effective guidance for the treatment of PTB or multidrug‐resistant tuberculosis (MDR‐TB).

Diagnostic Efficacy of Xpert MTB/RIF Assay in Bronchoalveolar Lavage Fluid for Tracheobronchial Tuberculosis: A Retrospective Analysis.

Background: The Xpert Mycobacterium tuberculosis/rifampin (MTB/RIF) assay has shown good diagnostic efficacy in brushing and biopsy tissue samples from patients with tracheobronchial tuberculosis (TBTB). However, its diagnostic value in bronchoalveolar lavage fluid (BALF) is still unclear. Therefore, the present retrospective study aimed to evaluate the diagnostic value of the Xpert MTB/RIF assay in BALF.Methods: The clinical data of 266 patients with suspected TBTB from January 2018 to October 2020 were pooled with complete details of bronchial brush and bronchoalveolar lavage samples. Smears of the bronchial brushings were stained with Auramine O stain to detect acid-fast bacilli (AFB), and BALF samples were used for culturing MTB with the BACTEC MGIT 960 system and the Xpert MTB/RIF assay. The diagnostic performance of these methods was assessed and compared.Results: A total of 266 patients suspected to have TBTB were enrolled in the final analysis. Of these patients, 179 patients were confirmed to have TBTB and 87 patients were non-TBTB. The sensitivity of the Xpert MTB/RIF assay in BALF (87.2%) was significantly higher than that of the brush smear for AFB (35.2%, p < 0.001). No significant difference was observed between the sensitivities of the Xpert MTB/RIF assay in BALF and MTB culture in BALF (87.2 vs. 84.9%, p = 0.542). The specificities of the Xpert MTB/RIF assay in BALF, MTB culture in BALF, and the bronchial brush smear were 97.7, 97.7, and 98.9%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of the Xpert MTB/RIF assay in BALF, MTB culture in BALF, and the bronchial brush smear were 98.7 and 78.7%, 98.7 and 75.9%, and 98.4 and 42.6%, respectively. Among the MTB culture-positive patients with TBTB detected by the Xpert assay, 27.0% (20/74) were identified to be resistant to RIF.Conclusions: The Xpert MTB/RIF assay in BALF enables a rapid and accurate diagnosis of TBTB and identification of RIF resistance, which is crucial for timely and proper treatment. Moreover, in patients with TBTB, BALF could be used as an alternative to bronchial brushing and biopsy tissues for the Xpert MTB/RIF assay.

Efficacy of the Xpert Mycobacterium tuberculosis/rifampicin assay for diagnosing sputum-smear negative or sputum-scarce pulmonary tuberculosis in bronchoalveolar lavage fluid

ObjectiveTo evaluate he diagnostic performance of the Xpert Mycobacterium tuberculosis/Rifampin (MTB/RIF) assay in bronchoalveolar lavage fluid (BALF). MethodsWe retrospectively reviewed the clinical data from 671 sputum-smear negative or sputum-scarce adult patients with suspected pulmonary tuberculosis (PTB) who had an Xpert MTB/RIF assay performed on BALF. The diagnostic performance of the Xpert MTB/RIF assay, smear microscopy (SM) and MTB culture was evaluated using MTB culture or final clinical diagnosis as the reference standard. ResultsCompared with MTB culture, the sensitivity and specificity were 87.8% and 72.7% for the Xpert MTB/RIF assay and 11.0% and 99.2% for SM, respectively. Compared with final diagnosis, diagnostic performance was 58.9% and 83.9% for the Xpert MTB/RIF assay, 5.0% and 98.3% for SM, and 43.3% and 100% for culture, for sensitivity and specificity respectively. The Xpert MTB/RIF assay had low specificity and high sensitivity. When very low results were re-evaluated and considered MTB-negative, the specificity increased significantly. The sensitivity remained higher than SM and was similar to that of culture. ConclusionsThe Xpert MTB/RIF assay adds microbiologic evidence to clinical decisions; however, close attention should be paid to very low semi-quantitative positive results.

Common bacteria in sputum or gastric lavage of patients presenting with signs and symptoms of lower respiratory tract infections.

Introductionlower respiratory tract infections (LRTIs) are infections involving the trachea, primary bronchi and lungs. People with LRTIs typically experience coughs as the primary symptoms; however, shortness of breath, weakness, fever and fatigue may be coupled with the cough. It is common among the aged, children under five and the immune-suppressed. Persons with symptoms suggestive of pulmonary tuberculosis (TB) may have tuberculosis, other respiratory tract infection or co-infection of tuberculosis and other respiratory pathogens. This study aimed to identify the presence of pathogens in sputum of suspected tuberculosis cases and their antimicrobial resistance patterns.Methodsthis was a retrospective study conducted from September 2018 to November 2019 at Tamale Public Health Laboratory. Sputum or gastric lavage samples were collected from persons with suspected clinical presentations of TB and/or LRTI. These samples were cultured using standard microbiological protocols and antimicrobial susceptibility test performed on the positive cultures by Kirby-Bauer disc diffusion method. Molecular identification of M. tuberculosis was performed on all the suspected TB cases using GeneXpert mycobacterium tuberculosis/rifampin (MTB/RIF) assay.Resultsduring the study period, there were 264 cases of which 49.2% were males and 50.8% were females. Positive cases for culture were 47.3%. Out of the 264 cases, 186 (70.5%) were suspected TB with 51.6% being positive for culture, 6.5% positive for M. tuberculosis (GeneXpert confirmed) and 3.8% co-infection of TB with other bacteria pathogens. Klebsiella spp. (35/125; 28%) and Pseudomonas spp. (19/125; 15.2%) were the most predominant pathogens isolated. There was no significant difference in detection of bacteria in males and females (p=0.89), however individuals with suspected TB were significantly infected with other bacterial species than the unsuspected individuals (p=0.03). Almost all the isolates showed high susceptibility towards carbapenem (meropenem) and high resistance towards the third generation cephalosporins (cefotaxime and ceftriaxone).Conclusionthis study highlights the need to test individuals with classical symptoms of LRTIs for other bacterial infections other than TB only. Sputum culture is recommended for all suspected tuberculosis cases to provide accurate laboratory diagnosis to LRTIs and mitigate unnecessary use of antimicrobials.

Utility of the GeneXpert Mycobacterium tuberculosis/Rifampin (MTB/RIF) Assay on Paraffin-Embedded Biopsy Tissue Samples for Detecting Tuberculosis: Comparison With Histopathology.

IntroductionThe Xpert Mycobacterium tuberculosis/Rifampin (MTB/RIF) assay is a qualitative nested real-time polymerase chain reaction (PCR) performed on the GeneXpert instrument system. Although, the utility of this technique for detecting tuberculosis (TB) in sputum and pus samples is well established, however, the usefulness of GeneXpert on biopsy samples is still a matter of debate. Therefore, in this study, we evaluated the utility of GeneXpert for detecting MTB in biopsy specimens diagnosed with TB.MethodsA retrospective observational study was conducted at the Department of Pathology, Liaquat National Hospital and Medical College. The data collection period was between January 2016 and December 2018 over a period of three years. Specimens included trucut/incisional biopsies and lymph node excisions. Cases with a favoured histopathological diagnosis of TB were included in the study. The Xpert MTB/RIF assay was performed on the samples obtained from paraffin-embedded biopsy tissue material, and comparison of histological features with Xpert MTB/RIF assay was performed.ResultsA total of 114 cases were included in the study. The mean age of the patients was 40.53±16.83 years, and 57.9% of patients were male. 68.4% of cases were extra-nodal with the lung being the most common extra-nodal site. On histopathological analysis, caseation necrosis, epithelioid granulomas and Langhan's giant cells were present in 64.9%, 70.2%, and 59.6% cases, respectively. On GeneXpert PCR assay, MTB was detected in 26.3% cases. A significant association of MTB detection on Xpert PCR assay was noted with the presence of necrosis on histopathology.ConclusionIn our study, we noted that the MTB detection rate by GeneXpert assay on histopathologically diagnosed cases of TB was only 26.3%, and the detection rate was significantly increased in the presence of caseation necrosis on a biopsy tissue sample. Despite the low detection rate due to rapid turnover time, GeneXpert assay is an excellent adjunctive tool for detecting MTB in paraffin-embedded tissue samples.

A Study on Diagnostic Evaluation of Two Different Rapid DNA Polymerase Chain Reaction Techniques Namely Gene Xpert Mycobacterium Tuberculosis/Rifampin (MTB/RIF) and Mycoreal Polymerase Chain Reaction in the Diagnosis of Endometrial Tuberculosis Considering Culture as Gold Standard.

Context:The study involves the evaluation of two polymerase chain reaction (PCR) techniques one of which has been endorsed by the WHO for their diagnostic capabilities.Aims:The aim of this study is to evaluate the diagnostic accuracy of GeneXpert mycobacterium tuberculosis/Rifampin (MTB/RIF) and mycoreal PCR techniques in the diagnosis of endometrial tuberculosis (TB) considering culture as the gold standard.Settings and Design:A retrospective study conducted at Gunasheela surgical and maternity hospital. Patients who attended the outpatient department between January 2013 and August 2016, satisfying the eligibility criteria, were included in the study.Methodology:Women included in the study underwent endometrial pipelle sampling premenstrually after ruling out pregnancy in that cycle. Endometrial samples were tested for TB by Mycoreal PCR, Gene Xpert and BACTEC culture.Statistical Analysis Used:Statistical analysis was done using the R software version 3.6.1. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of test were calculated.Results:A total of 3229 samples were analyzed, of which 1754 were evaluated by Mycoreal TB PCR and 1475 were evaluated by Gene Xpert MTB/RIF assay. The sensitivity of mycoreal TB PCR technique was 34.78%, specificity was 99.08%, PPV was 33.33%, NPV was 99.13%, and accuracy was 98.23%. The sensitivity of GeneXpert MTB/RIF technique was 6.90%, specificity was 99.79%, PPV was 40.00%, NPV was 98.16%, and accuracy was 97.97%.Conclusions:MYCOREAL seemed to be more sensitive than Gene Xpert (MTB/RIF) considering culture as the gold standard in the diagnosis of endometrial TB.

Possible Binding of Piperine in Mycobacterium tuberculosis RNA Polymerase and Rifampin Synergism.

The activity of rifampin (RIF) and piperine was evaluated at the relative transcript levels of 12 efflux pumps (EPs), and an additional mechanism was proposed to be behind the synergic interactions of piperine plus RIF in Mycobacterium tuberculosis AutoDock v4.2.3 and Molegro v6 programs were used to evaluate PIP binding in M. tuberculosis RNA polymerase (RNAP). A hypothesis has been raised that piperine interferes in M. tuberculosis growth through RNAP inhibition, differently from what was previously endorsed for EP inhibition only.

Evaluation of Xpert®Mycobacterium tuberculosis/rifampin in sputum-smear negative and sputum-scarce patients with pulmonary tuberculosis using bronchoalveolar lavage fluid.

Context:Sputum smear-negative and sputum-scarce pulmonary tuberculosis (PTB) is a diagnostic challenge. Xpert® Mycobacterium tuberculosis/rifampin (MTB/RIF) provides a rapid diagnosis on high-quality biological specimen obtained by bronchoscopy.Aims:The aim of this study is to evaluate Xpert® MTB/RIF on bronchoalveolar lavage (BAL) fluid in sputum smear-negative and sputum-scarce PTB patients.Settings:Tertiary care hospital in India.Design:This was prospective observational study.Materials and Methods:Between January 2015 and November 2016, we prospectively recruited sputum-smear negative and sputum-scarce patients under evaluation for PTB and performed BAL. Sensitivity, specificity, positive, and negative predictive values were calculated for the diagnosis of PTB on BAL fluid for acid-fast bacilli smear and Xpert® MTB/RIF using liquid culture as the reference standard and compared to the final diagnosis based on composite reference standard. Sensitivity, specificity, and predictive values were calculated with 95% class intervals. McNemar's test was used for comparison of sensitivities.Results:Of the 60 patients included, 52 (88.3%) had a final diagnosis of PTB and 16 (26.7%) were culture confirmed. Xpert® MTB/RIF had a sensitivity and specificity of 81% (54%–96%) and 73% (56%–85%) in culture confirmed cases; 46% (32%–60%) and 100% (63%–100%) for the final diagnosis; 32% (17%–51%) and 100% (54%–100%) in culture negative cases, respectively. Culture had a sensitivity of 32% (20%–47%) for the final diagnosis.Conclusions:In sputum smear-negative and sputum-scarce patients with clinico-radiological features of PTB Xpert® MTB/RIF has good sensitivity for diagnosis on BAL fluid. It is useful even when cultures are negative.

Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based drug susceptibility testing (DST).We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay.Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 µg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 µg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation.Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays.

Evaluation of Genotype® MTBDRplus Assay for Rapid Detection of Resistance to Isoniazid and Rifampin in Mycobacterium tuberculosis Isolates Collected in Tunisia

TB diagnosis and resistance detection remain to suffer from complex techniques and long time taking. The rapid identification of drug-resistant strains of Mycobacterium tuberculosis is crucial for the timely initiation of appropriate antituberculosis therapy. Over the years, several new technologies have been proposed to accelerate and simplify the detection of resistant M. tuberculosis. In this context, we evaluated the Genotype MTBDRplus to detect resistance to isoniazid and rifampin in positive M. tuberculosis cultures. The performance of the Genotype MTBDRplus assay was compared with that of the phenotypic drug susceptibility tests, a gold standard culture based method. The Genotype MTBDRplus assay was quicker and more cost-effective for the detection of rifampin and isoniazid resistance, with a slightly lower detection of rifampin-resistant strains in our setting.

Evaluation of the CombiChip Mycobacteria™ Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis

The CombiChip Mycobacteria™ Drug-Resistance Detection DNA chip, recently developed by GeneIn (Pusan, South Korea), is an oligonucleotide microchip coupled with polymerase chain reaction for the detection of mutations associated with resistance to isoniazid (INH) and rifampin (RIF). This oligonucleotide chip was compared with DNA sequencing and phenotypic drug susceptibility testing with 69 INH- and/or RIF-resistant and 27 all tested drug-susceptible Mycobacterium tuberculosis isolates. Two selected codons (the katG codon 315 and inhA15) allowed identification of 84.1% of INH-resistant isolates and 100% of RIF resistance were detected by screening for 7 codons: rpoB511, rpoB513, rpoB516, rpoB522, rpoB526, rpoB531, and rpoB533. The overall specificity of this oligonucleotide chip for detecting INH and RIF resistance were 100 and 95.3%, respectively. This level of sensitivity and specificity is concordant with that from the determination of M. tuberculosis drug resistance by DNA sequencing. This oligonucleotide chip is a rapid and reliable genotypic method capable of detecting multiple mutations associated with INH and RIF resistance simultaneously in a single microchip slide.

Detection of Mutations in RNA Polymerase Beta Subunit Gene Encoding Resistance to Rifampin in Mycobacterium tuberculosis by DNA Microarray

Rapid and efficient diagnosis is essential in the management of drug‐resistant tuberculosis. A DNA microarray technique based on differential hybridization method was described in the present study for detecting mutations in the RNA polymerase beta subunit (rpoB) gene of Mycobacterium tuberculosis (M. tuberculosis) cultures and in clinical specimens. The mutations in rpoB confer resistance to rifampin, an important first‐line antituberculosis drug. The differential hybridization approach was mainly based on the effect of a single base mismatch on the melting temperature of the hybridized DNA; therefore, any point mutation of rpoB gene resulting in the rifampin resistance can be detected efficiently. The development of the DNA microarray involves the design of dozens of oligonucleotide probes for identifying rifampin‐resistant and ‐sensitive strains. The method comprises isolating genomic DNA from the samples containing M. tuberculosis cells, amplifying rpoB gene coding sequence to produce fluorescently labelled product, and hybridization with the oligonucleotide arrays. The results demonstrated the capability of DNA microarray to provide important clinically relevant information about the rpoB gene of mycobacterial organisms. The DNA microarray offers a reliable diagnostic test for rapidly detecting multidrug resistance caused by gene mutations of mycobacteria.

Characterization of rifampin-resistance in pathogenic mycobacteria.

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.